Resistance of insect pests to insecticides is a major problem worldwide and has appeared in over 170 arthropod species of public health importance. Increased detoxification of insecticides by cytochrome P450 enzymes is a major mechanism of resistance, but its molecular basis is unknown. The long term objective of this proposal is to study the structure, function and regulation of insect cytochrome P450 genes and proteins. This basic knowledge is needed to understand the molecular basis of insecticide resistance in insects as well as the diversity and evolution of P450 genes. This knowledge will be needed in resistance management programs which are becoming a part of integrated pest management systems. The proposal focuses on insecticide-resistant and susceptible strains of the house fly, Musca domestica, and will continue the studies which have already yielded cDNAs clones and sequences for a cytochrome P450 and for NADPH:cytochrome P450 reductase. The specific aims of this proposal are:(l) To elucidate the structure of P450 genes and proteins: The effort to clone and sequence P450 cDNAs will be continued in order to characterize those P450s involved in insecticide resistance and to document the multiplicity of P450 in insects. This effort will be supported in part by a new technique to obtain probes for P450 proteins based on photoaffinity labeling. The sequences of P450 proteins and the intron-exon organization of the P450 genes will be compared and their evolutionary relationships will be studied. (2) To define the function of P450 proteins: Polyclonal antibodies to P450 polypeptides expressed in bacteria and to P450 peptide segments will be raised. An expression system for P450 proteins and fusion proteins of P450 reductase and cytochrome P450 in yeast will be developed. Inhibition of microsomal P450 activity by antibodies and assays of P450 expressed in yeast will be used to define what reactions are catalyzed by individual P450 proteins. (3) To study the regulation of P450 gene expression and the molecular basis of P450-mediated insecticide resistance: The hypothesis that insecticide resistance in the Rutgers strain is caused by an altered (increased) P450 gene expression will be tested. Evidence for the existence of trans-acting factor(s) will be sought and trans-regulation of P450 gene expression will be investigated.